I. Molecular architecture of multi-protein enzyme complexes: We have been using cryo-electron microscopy to study the architecture of the pyruvate dehydrogenase complex as an example of a multi-enzyme complex involved in energy metabolism. So far, we have determined the three dimensional structures at 27 resolution for two giant icosahedral pyruvate dehydrogenase complexes from Bacillus stearothermophilus using electron cryo-microscopy: one is a 11 MDa complex composed of 60 copies of E1 and E2 enzymes, and the other is a 9 MDa complex composed of 60 copies of E2 and E3 enzymes. By positioning the previously determined structures of E1, E3 and the three domains of E2 into the model, we have arrived at atomic interpretations for the entire E1E2 and E2E3 complexes. To our knowledge, these are the largest non-viral protein complexes for which such atomic models are available, and they provide unique insights into the functional mechanism of a fascinating cellular machine that remains inaccessible to structural analysis by X-ray crystallography. In ongoing work, we are studying the role of flexible linker regions in maintaining the size and shape of the complex using a combination of electron microscopic and other biophysical approaches. Studies of mammalian pyruvate dehydrogenase complexes to understand the mechanisms underlying regulation by kinases and phosphatases are also planned. II. Spatial map of molecular complexes in healthy and diseased mitochondria We are using Dictyostelium discoideum, a genetically amenable haploid model system that undergoes a defined program of cellular differentiation and morphogenesis, to develop methods to determine the molecular architecture of mitochondria. Dictyostelium mitochodria are small ( 0.5-0.7 micrometer in diameter and 0.5-2.0 micrometer in length) and easily isolated, and can be imaged using electron tomography. We have established the feasibility of this imaging approach in our recent work on electron tomography of an intact microorganism Bdellovibrio bacteriovorus, where we have demonstrated that it is possible to visualize individual molecular machines in the context of an intact cell. In related experiments, we are imaging whole Dictyostelium cells using a newly developed 3D imaging tool known as dual beam microscopy to map the spatial distribution of mitochondria in cells at different stages of development, and in mitochondrial mutants. We expect these studies will be relevant for the exploration of structural aspects of mitochondrial defects in cancer.